Our investigation aims to establish how the atomic and membrane-level events operate to trigger visual signal transduction by rhodopsin in a unified multi-scale framework, with broad implications for biological signaling. The high impact of understanding G-protein-coupled receptor (GPCR) activation is well appreciated. Yet numerous gaps in our understanding remain, both with rhodopsin, as well as other Family A GPCRs for which rhodopsin is a highly significant prototype. Here we plan to resolve the long sought, critical mechanistic features by an innovative approach that combines magnetic resonance (solid-state 2H and 13C NMR), Fourier transform infrared (FTIR), and electronic (UV-visible) spectroscopy. Our novel hypothesis for rhodopsin activation is formulated in terms of factors that drive a progression of transient conformational substates through an active ensemble: release of retinal strain, retinal-specific protein dynamics, hydration changes, pH catalysis, dynamical G-protein coupling, and membrane stress due to the polyunsaturated lipid composition. (1) Application of our novel solid-state 2H and 13C NMR technology will reveal how the release of conformational strain through retinal isomerization and relaxation unlocks the active rhodopsin (Meta-II) state. The angular and distance restraints from 2H and 13C solid-state NMR will illuminate the dynamical structure of retinal through its progression of active sub-states toward the active Meta-II form. (2) Changes in local retinal dynamics as studied by 2H and 13C NMR relaxation studies will be correlated with rhodopsin's activating motions by combining the results of spectroscopy with molecular dynamics (MD) simulations. Local retinal mobility will be related to large-scale protein dynamics involving fluctuations of the transmembrane helices. Notably, this work will address ambiguous X-ray structural data with results obtained at more physiological temperatures. Our investigation will decide the question of why active Meta-II rhodopsin and the ligand-free Opsin* apoprotein have similar X-ray structures, yet completely different activities. (3) Next we plan to investigate the specific role f pH and hydration throughout the active ensemble in relation to rhodopsin's interaction with transducin. We plan to combine our spectroscopic methods (UV-visible, FTIR, site-directed spin-labeling) with osmotic pressure studies to investigate changes in water-mediated H-bonding networks, together with a dramatic water influx due to transmembrane helical movements in rhodopsin activation. (4) Additional research will uncover the influences of polyunsaturated lipids on rhodopsin through modulation of membrane curvature stress, and how lipid composition biases the distribution of states in the active ensemble mechanism. Our plan encapsulates a new multi-scale view of how rhodopsin initiates visual signal transduction, tying together mechanical, environmental, temporal, and structural factors. Understanding how these factors interoperate across various length and time scales is fundamental to understanding of how rhodopsin achieves the extreme high fidelity required for visual signaling. Our robust and novel approach will provide important insights that are transferable to the broader class of GPCRs in biology and pharmacology.